Possible involvement of zinc transporter ZIP13 in myogenic differentiation

Ehlers–Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.

hiPSCs MYOD were dissociated into single cells using a detachment solution and seeded on Matrigel-coated 24-well plate in StemFit medium supplemented with 10 µM Y27632.
The culture medium was replaced the next day with primate ES cell medium.On Day 2, 1 µg/mL DOX was added to the same medium, and the latter was replaced with minimum essential medium eagle, alpha modification (MEM) supplemented with 5% knockout serum replacement (KSR), 200 µM 2-mercaptoethanol (2-ME), and 1 µg/mL DOX on Day 3.This myogenic differentiation was performed at 37 °C in the presence of 5% CO2 until Day 8. (b-g) Differentiation of iPSCs MYOD from healthy controls (H1-iPSCs MYOD and H2-iPSCs MYOD ) and patients with EDSSPD3 (EDSSPD3-P1-iPSCs MYOD and EDSSPD3-P2-iPSCs MYOD ) into myocytes was induced by hMyoD overexpression under the control of DOX, as s according to Supplementary Figure S5a.The experiments of myogenic differentiation using H1-iPSCs MYOD and EDSSPD3-P1-iPSCs MYOD or H2-iPSCs MYOD and EDSSPD3-P2-iPSCs MYOD were performed simultaneously, respectively.EDSSPD3, EDSSPD3-P1-iPSCs MYOD (n = 9) and EDSSPD3-P2-iPSCs MYOD (n = 9), on Days 3, 6, and 8 were calculated with raw data in Figure 3d, e, g, and h and Supplementary Figure S6f and g, normalized to those of human GAPDH and expressed relative to the levels in H1-iPSCs MYOD on Day 3 (set as 1).Simultaneously, we statistically analyzed the difference in these expression levels between the averaged data across three healthy controls or two patients with EDSSPD3 using Student's t-test.Data are presented as mean ± SEM. ***P < 0.001 compared to the indicated groups for healthy controls iPSCs MYOD .

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into myocytes were induced with Dulbecco's modified eagle medium (DMEM) containing 2% horse serum (HS) as depicted in Figure 1a.(a) Microscopic evaluation of the morphological changes in Scramble clones of C2C12 cells (clone number: 5, 9, and 13) during myogenic differentiation on Days 0 and 3. Scale bars, 150 μm.(b-d) Gene expression profiles of HS-induced mZip13 (b) and myogenic differentiation markers, mMyoD (c) and mMyogenin (d), (n = 3 each) analyzed by qPCR.The cells were harvested at the indicated time points.Data are presented as the mean ± standard error of mean (SEM) of three independent experiments.*P < 0.05 and **P < 0.01 relative to the data on Day 0. morphological changes in Scramble 9 and Zip13-KD 6 of C2C12 cells at the indicated time points after myogenic differentiation stimuli with DMEM containing 2% HS.Scale bars, 150 μm.(b) Downscaled microscopic images of Supplementary Figure S3a were shown.Scale bars, 300 μm.Supplementary Figure S4.Sequencing analysis of ZIP13-exon2 mRNA from iPSCs derived from patients with EDSSPD3 harboring ZIP13 G64D mutation.Total RNA was extracted from the lysates of dermal fibroblasts and iPSCs from healthy controls (H1 and H2) or patients with EDSSPD3 harboring ZIP13 G64D mutation (EDSSPD3-P1 and EDSSPD3-P2), and used to synthesize cDNA.The synthesized cDNA was used as a template for PCR using human ZIP13-exon2 mRNA primers.Their PCR amplicons were analyzed by Sanger sequencing.Codon GGT: Glycine (Gly), GAT: Aspartic acid (Asp).
Figure S5a.The experiments of myogenic differentiation using H1-iPSCs MYOD and EDSSPD3-P1-iPSCs MYOD or H2-iPSCs MYOD and EDSSPD3-P2-iPSCs MYOD were performed simultaneously, respectively.(a-o) The relative mRNA expression analysis for skeletal muscle or myogenic precursor markers in differentiated H1-iPSCs MYOD and EDSSPD3-P1-iPSCs MYOD or H2-iPSCs MYOD and EDSSPD3-P2-iPSCs MYOD .The mRNA expression levels of zinc status marker, hMT1H (a and h) (n = 9) was determined by RT-qPCR on Days 0, 3, 6, and 8, normalized to that of human GAPDH and expressed relative to the levels in healthy-iPSCs MYOD on Day 0 (set as 1).The mRNA expression levels of skeletal muscle markers, hMYH (b and i), hGLUT4 (c and j), and hCK-M (d and k) and myogenic precursor markers, hMYF (e and l), hPAX3 (f and m), and hPAX7 (g and o) (n = 9 each) were determined by RT-qPCR on Days 3, 6, and 8, normalized to that of human GAPDH and expressed relative to the levels in healthy-iPSCs MYOD on Day 3 (set as 1).Data are presented as mean ± SEM and are representative of three independent experiments.*P < 0.05, **P < 0.01, and ***P < 0.001, versus the indicated groups for healthy-iPSCs MYOD .***P < 0.001 versus healthy-iPSCs MYOD on Day 0 (a and h).† † † P < 0.001 versus EDSSPD3-patients-iPSCs MYOD on Day 0 (a and h).(p-s) The protein expression levels of hMYH in cell lysates were analyzed by western blotting on Day 8 (p and r).β-ACTIN protein was used as an internal control.Signal intensities were measured using ImageJ software.The protein levels of hMYH (q and s) (n = 5 each) were calculated by normalizing them to that of β-ACTIN and expressed relative to the levels in healthy-iPSCs MYOD on Day 8 (set as 1).Data are presented as mean ± SEM and are representative of five independent experiments.***P < 0.001 versus healthy-iPSCs MYOD .
RC3 on Day 0. (d and e) Relative protein expression analysis for hMYOGENIN in differentiated EDSSPD3-P1-iPSCs MYOD or RC1-3.The protein expression level of hMYOGENIN in cell lysates were analyzed by western blotting on Day 8 (d).β-ACTIN was used as an internal control.Signal intensities were measured using ImageJ software.The protein expression level of hMYOGENIN (n = 30 each) were calculated after normalizing them to that of β-ACTIN and expressed relative to the levels in EDSSPD3-P1-iPSCs MYOD on Day 8 (set as 1) (e).Data represent the mean ± SEM and are representative of ten independent experiments.**P < 0.01 versus EDSSPD3-P1-iPSCs MYOD .presented in Figure 1k.Uncropped full-length images of western blotting membrane of MYH protein expression in Scramble 1 and two clones of Zip13-KD (clone 6 and 7) of C2C12 cells during myogenic differentiation on Days 0 and 3. Black arrows showed protein bands recognized by indicated antibodies, and these bands are presented in Figure 1k.